Toaddressacriticalneedforimprovedhumantissuemodelstostudyinfectiousdiseases,weproposetoform theMITCenterforHumanTissuesandInfectiousDiseases(MIT.HTMID).TheMIT.HTMIDCenterwillstudy viralinfectionsofthehumanbrainandcentralnervoussystembyusingbiologicallyrelevanttwodimensional (2D)humanneuralcellsandthreedimensional(3D)humancerebralorganoids.Thecellsarehomogeneous preparationsofhumanneuronalprogenitors,neurons,oligodendrocytes,astrocytes,andmicroglia,derived fromembryonicstem(ES)cellsandinducedpluripotentstemcells(iPScells),andproducedintheHumanCell andTissueCore.Priorworkhasdemonstratedthatthesecellsandtheorganoidsformedare nearphysiologicalintheirbiologicalfunctions,therebyrepresentinganexperimentalsystemthatissuperiorto rodentmodels.ThegoalofProject1,Aim1Aistodefinetheviralinfectionphenotypesforfivecelltypes.Two typesofviruses,producedintheMIT.HTMIDVirologyCore,willbeused.First,wewillstudyflaviviruses, includingDengueFeverVirus,WestNileVirus,andZikaVirus.Indeed,animportantmotivationforstudying humanbraintissueandvirusesistounderstandthepathogenesisofZikavirusinfections,whicharecausing microcephalyandGuillainBarresyndromeworldwide.Second,wewillusepseudotypedvesicularstomatitis viruses(VSV)tostudyentryofseveralencephaliticviruses,includingEasternEquineEncephalitisVirus (EEEV),WesternEquineEncephalitisVirus(WEEV)andVenezuelanEquineEncephalitisVirus(VEEV).By replacingtheVSVviralenvelopewiththatofEEEV,WEEV,orVEEV,weareabletostudyvirusentryinthe2D cellsand3Dorganoids.TheseexperimentsarehighlysignificantbecauseEEEV,VEEV,andWEEVare selectagents.Usingpseudotypedselectvirusesandmultiplerelevantcelltypeswillallowustostudyvirus tropism,pathology,andpotentialtherapeutictargets.Project1Aim1Awillexaminevirusinfectionphenotypes? thatis,wewillanalyzevirusreplication,cellviability,spontaneouselectricalactivity,transcriptomeand secretomechanges,andpotentialtodifferentiatetoothercelltypes.Thisapproachissignificantbecauseitwill demonstratewhichcelltypesaremostsusceptibletovirusinfections,alsorevealingsimilaritiesanddifferences amongvirusesthatcauseverydifferentclinicaldiseases.InProject1,Aim1B,weproposetostudyvirus infectioninthecontextofthethreedimensionalhumancerebralorganoidtissuemodel.Ourgroupmaybe uniqueinhavingthetechnicalcapacitytogenerateorganoidsthatincludemicroglia,whicharetheimmune cellsofthebrain.Ourgoalwillbetodetermineifactivatedmicrogliahaveprotectiverolesbysecretingfactors thatestablishanantiviralenvironment.Alternatively,wewillalsotestahypothesisthatmicroglia,upon infectionwithZikavirus,areabletomigrateintocerebralorganoidstoinitiatecytotoxicinfectionofneuralcell types.TheseexperimentsareaimedatunderstandinghowthefetalbrainbecomesinfectedbyZikavirusto causemicrocephaly.TheworkwillbefacilitatedgreatlybyformingtheMIT.HTMIDCenter.